The proposed research consists of two sections, the chemistry of the amatoxins with respect to their specific complexation with eukaryotic RNA polymerases and the application of selected derivatives to study the transcriptive functions of eukaryotic RNA polymerase. Examination of the amatoxin chemistry will focus upon the hydroxyindole moiety of alpha-amanitin. The purified derivatives are to be tested for their inhibition properties on eukaryotic RNA polymerases I, II, and III. Those derivatives with Ki less than 10 to the minus 6th power M for RNA polymerase III or II are to be radiolabelled and characterized with respect to KD and half-life of the amatoxin-RNA polymerase complex. Structural assignments for each derivative are to be obtained by comparison with previously defined compounds and by proton magnetic resonance. The objective of this research is to obtain high specific radioactivity derivatives suitable for study of RNA polymerase II and III in ligand binding experiments. The radioactively tagged amatoxin will provide a ligand binding assay to quantitate the association and dissociation of RNA polymerase II with DNA and with chromatin fragments. This will permit an estimation of the affinity for the RNA polymerase-DNA complex. The effect of DNA structure (e.g. double-stranded and single-stranded DNA as well as nuclease treated) on the kinetic parameters will be established.